PROJECT 3 (TISSUE MODELING PROJECT) - PROJECT SUMMARY A successful microbicide product has pharmacokinetics (PK) that effect prophylactic pharmacodynamics (PD). Product design should manipulate candidate compositions, volumes, and drug packaging in a rational manner to yield target PK/PD; but we have limited understanding of this relationship. Further, our methods for experimentally evaluating PK in animals and humans are limited: they do not delineate drug concentration distributions throughout target compartments where the drugs act, and may miss drug partitioning and concentration gradients that drive drug transport; they fail to measure key molecules, e.g. drug metabolites, HIV, and intracellular molecules that might modulate drug efficacy (e.g. endogenous nucleotides). Thus, drug concentrations in contemporary PK studies, while useful, do not adequately inform us about product PK and may not accurately reflect true prophylactic potential. Project 3 will develop and apply new methodology - experimental and computational - to understand and predict these critical, heretofore missing elements of PK, for the enema microbicide product, as it is being designed and evaluated by the DREAM Program. Our team has synergistic expertise in both experimental methods and computational modeling of the kind needed here. Aims 1 and 3 develop this unprecedented, transformative methodology. Aim 1 integrates detailed MALDI measurements of drug, metabolite and other key molecules in colorectal tissue specimens with those by a specialized instrument that applies confocal Raman spectroscopy - to measure local microbicide drug concentrations in tissue specimens in 3D - coupled to spectral domain optical coherence tomography - to link drug concentration to structures of the mucosal tissue within which drug is migrating. Aim 3 creates a new biophysics and physiology based computational compartmental model of detailed PK for enema designs. It predicts the 3D, time-dependent drug concentration distributions in these compartments - delineating PK with greater resolution than previously possible. Aims 2 and 4 and apply the methods of Aims 1 and 3 to the human studies in Project 1 and 4 and NHP studies in Project 2, helping interpret PK and PD data, and thence understand, design and choose the best microbicide enema designs.